We have found that motoneuron-differentiated ES cells survive when transplanted into adult rats with a motoneuron injury and that many of the cells exhibit immunohistochemical features of mature motoneurons. In this project, we will determine strategies that facilitate the generation of functional ES cell-derived motoneurons in the injured adult, mammalian CNS. In specific aim 1, we will define host-applied and ex vivo strategies that optimize motoneuron differentiation and survival in the acutely damaged spinal cord. We will determine the ability of these cells to survive and engraft within the rat spinal cord at each of several differentiation stages (i.e. pluripotent stem cell, committed neural precursor, committed motoneuron precursor or postmitotic motoneuron). Additionally, we will explore whether the environment into which the cells are transplanted modulates survival and differentiation. In specific aim 2, we will test approaches that enhance the ability of ES cell-derived axons to overcome CNS white matter-mediated axonal repulsion. We will modulate the activity of NOGO-R, P75-NTR and RhoA in order to transiently render the transplanted ES cells less susceptible to host repulsive cues as they navigate surrounding white matter. In specific aim 3, we will test approaches that enhance the ability of ES cell-derived motoneurons to extend axons distally and allow the formation of functional neuromuscular junctions in vivo. Based on expression of neurotrophin receptors on ES-derived motoneurons, we propose to peripherally express cognate ligands (including IGF-1 and GDNF) that will attract ES-cell derived motor axons to grow distally and to innervate muscle. Retrograde labeling, electrophysiologic testing and behavioral studies will determine the effectiveness of these strategies in generating functional motoneurons and restoring motor units in paralyzed animals. These studies will define an important transplantation approach for motoneuron disease such as ALS and SMA and for spinal cord injury and other spinal disorders.